Description Although tremendous progress has been made in primary and secondary prevention, the complications of atherosclerosis (heart attacks, strokes and peripheral artery disease) still account for the most deaths worldwide. Here, we propose to test three hypotheses. This work has become possible through the intravital live cell triggered imaging system (ILTIS) we developed in the current funding cycle. Hypothesis 1 is that non- classical monocytes use LFA-1 (?L?2 integrin) and ~40% also VLA-4 (?4?1 integrin) to patrol the endothelium overlying atherosclerotic plaque. We have preliminary evidence for at least two subsets of patrolling monocytes in atherosclerotic arteries. To test the molecular mechanism of patrolling, we will block integrins, their ligands, chemokines and chemokine receptors using monoclonal antibodies. Hypothesis 2 is that blood monocytes gain access to atherosclerotic plaque through the luminal endothelium. To test how monocytes enter atherosclerotic lesions, we have made mice with yellow (YFP) endothelium that are crossed into the Apoe-/- atherosclerosis- prone background. Combining these mice with mice in which monocyte subsets are fluorescently labeled (Cx3cr1GFP/+ for non-classical, Ccr2RFP/+ for classical monocytes) will show transendothelial migration of monocytes into atherosclerotic plaque in vivo. Hypothesis 3 is that four newly discovered plaque macrophage subsets (by ILTIS) have different functions. Preliminary RNA-Seq data show that their gene expression profiles vary significantly. We will test these functions: proliferation by BrdU and Ki67; oxLDL and minimally modified (mm)LDL uptake; efferocytosis; migration and antigen presentation. When the proposed work is complete, we will know, for the first time, how monocytes patrol atherosclerotic arteries, how they enter atherosclerotic lesions and what they do there. We will identify key functions of the four newly discovered monocyte and macrophage subsets in atherosclerotic plaque.